The present invention relates to the preservation of viruses and mycoplasma. In particular, it relates to an ultra-rapid method by which such materials can be preserved using the disaccharide, trehalose. By this method, a long term preservation of viruses and/or mycoplasma can be achieved and, especially, living attenuated vaccines can be prepared.
The preservation of biodegradable materials by dehydration and osmoconcentration is a familiar and ancient technology. When the task of preserving sensitive biomolecules became necessary, simple drying by dehydration failed, as structural water was removed, causing subsequent denaturation and loss of vital activity. Cryopreservation in liquid nitrogen and lyophilisation have become the accepted methods for the long term preservation of sensitive biomolecules, the latter method being used extensively for the preservation of live attenuated vaccines.
Improved thermotolerance of freeze dried Rinderpest vaccine has been achieved by extending the secondary drying cycle, in order to reduce residual moisture (RM) levels to around 1%-2%. This entails long and high energy consuming operational cycles of up to 72 hours as described by Mariner, J. C. et al., Vet. Microbiol., 1990, 21, 195-209. Vaccines produced by this method are known and are distinguished from the standard vaccine by the name xe2x80x9cTHERMOVAXxe2x80x9d.
As mentioned above, these currently used processes are time consuming and involve high energy input. Furthermore, lyophilisation confers only a modest level of thermotolerance in the final product and refrigeration is still required to reduce deterioration during storage. This is a particular problem for live vaccines to be used in tropical climates since these lose potency with the unfortunate result that vaccination programs carried out in the field in tropical countries, where monitoring the xe2x80x9ccold chainxe2x80x9d is difficult, ultimately lead to vaccination of patients with substandard or, in some cases, useless vaccine.
During evolutionary natural selection, certain species of plants and animals acquired the remarkable and elegant ability to tolerate extreme dehydration, remaining dormant in hostile environments for very long periods of time and yet able to assume complete vital activity on rehydration. Examples include the resurrection plant Selaginella lepidophya, the brine shrimp Artemia salina (Clegg, J., J.Comp.Biochem.Physiol, 1967, 20, 801-809), the yeast Saccharomvces cerevisiae (Coutinho, E., Journal of Biotechnology, 1988, 7, 23-32) and the tardigrade Macrobiotus hufelandi (Kinchin, I. M., Biologist, 1995, 42, 4). Such organisms are termed cryptobiotic and the process by which they survive is known as anhydrobiosis. All species of animals and plants which display this ability contain the disaccharide trehalose (xcex1-D-glucopyranosyl-xcex1-D-glucopyranoside). Its presence generally in the order of 0.2 g/g dry cell weight in most cryptobionts enables them to resist extreme dehydration, high temperatures, X-rays and also in some species of tardigrades, pressures as high as 600 Mpa.
Colaco et al., Biotechnology, 1992, 10, 1007-1011, describe the benefit of rapid drying of biological materials using trehalose. This method mostly refers to the drying of restriction enzymes and immunoglobulins onto preformed solid matrices, such as cellulose fibres or onto the surfaces of plastic plates for diagnostic purposes such as ELISA or similar diagnostic applications in the laboratory. Problems arise, however, with these techniques when scaling up to industrial applications, such as large scale commercial vaccine production where much larger unit numbers and volumes have to be handled using mandatory aseptic techniques in partially sealed vials. To meet the operational requirements of large scale vaccine production, where unit volumes from 1.0 ml upwards and production batches of 20 litres are typical, a different strategy is required to remove that volume of water in an economically acceptable time. Drying at atmospheric pressure even at the highest physiologically tolerated temperatures would require an unacceptably long time to remove water quickly enough from partially stoppered vaccine vials and would inevitably result in denaturation and loss of potency.
The present invention is concerned with a method of preservation of viruses or mycoplasma using trehalose under conditions which cause water to be removed while, at the same time, allew the biological integrity of the material to be maintained.
Accordingly, the present invention provides a method of preserving a biologically-active material comprising a live virus or mycoplasma which method comprises the steps:
(i) mixing an aqueous suspension of the biologically-active material with a sterile aqueous solution of trehalose to give a trehalose concentration in the mixture in the range of from 0.2 to 10% w/v;
(ii) subjecting the mixture to primary drying, for 30 to 60 minutes, at a pressure of less than atmospheric and at a temperature initially no greater than 37xc2x0 C., and which is controlled not to fall to 0xc2x0 C. or below and which finally is no greater than 40xc2x0 C. to form a glassy porous matrix comprising glassy trehalose having a residual moisture content of not greater than 10% and containing, within the matrix, desiccated biologically-active material; and
(iii) subjecting the glassy porous matrix of step (ii) to secondary drying for 10 to 30 hours at a pressure not greater than 0.1 mbar and at a temperature which finally is in the range of from 40 to 45xc2x0 C. to form a trehalose matrix having a residual moisture content of not greater than 2% containing, within the matrix, desiccated biologically-active material.
By using the method of the invention it is possible to produce a live vaccine with, compared to prior art methods, enhanced biological characteristics and distinct commercial advantages. Vaccines prepared using the method of the invention are dried much more quickly than those using conventional freeze drying procedures. For instance, the method of the invention can be used to dry trehalose/biologically-active material mixtures to a moisture content of about 10% in less than one hour. Further dehydration to a residual moisture content of about 1-2% can be achieved in less than 30 hours, for instance about 20 hours, compared to a period of 50 hours by conventional freeze drying procedures. Furthermore, damage caused by solute concentration is minimised according to the present invention and particularly damaging ice crystallisation is avoided. The thermostability of the biologically-active material preserved in the trehalose glassy matrix is greater than that of materials preserved by prior art methods and, thus, the necessity of the xe2x80x9ccold chainxe2x80x9d, which is a serious constraint with conventional freeze-dried vaccine, is minimised. The product of the present invention can be exposed to high ambient temperatures, e.g., up to about 45xc2x0 C., for prolonged periods without any substantial loss of biological activity. In addition to these and other advantages of the present invention the product of the method exhibits instantaneous xe2x80x9cflash solubilityxe2x80x9d upon rehydration.
The method of the present invention is suitable for achieving the long term preservation of viruses and mycoplasma. In particular, it can be used to preserve highly labile live attenuated viral components and mycoplasma components that can be rehydrated to form vaccines. Examples of such biologically-active materials that can be preserved according to the method of the invention include:
Family: Paramyxoviridiae
Subfamily: Paramyxovirinae
Genera: Parainfluenza virus group Measles, Rinderpest, canine
distemper, Peste des Petits Ruminants (PPR)
Paramyxovirus: mumps virus (Mumps)
Genus: Rubivirus, Rubella (German Measles)
Genus: Flavivirus, Yellow fever virus (Yellow Fever)
Genus: Rhabdoviruses, Lyssaviridiae (Rabies virus)
Picoma viruses (Polio virus)
Newcastle Disease virus
Mycoplasma: Mycoplasma mycoides (Contagious Bovine Pleuropneumonia)
Brucella abortus: Strain 19 vaccine
Chlamydia: Chlamydia psittaci (Enzootic abortion)
Coccidia: Toxoplasma gondii (Toxoplasmosis)
According to the method of the present invention, the biologically-active material to be preserved is prepared as a suspension in an appropriate aqueous medium. In the case of a virus, it may be that this will need to be cultured, for instance in vero cells, in an appropriate culture medium, and then harvested prior to suspension in order to provide a useful concentration of material. Typically, the aqueous suspension of biologically-active material will be pH adjusted, for example by the addition of an alkali, to a pH in the range of from 7.0 to 7.8 especially about 7.4.
Trehalose is one of the most stable and chemically non-reactive disaccharides. It has an extremely low bond energy of less than 1Kcal/Mol making the dimer structure very stable. It does not undergo caramelisation unless heated severely, nor does it cause the Maillard reaction with proteins or peptides. The natural di-hydrate structure containing two water molecules enables unusual flexibility around the disaccharide bond which possibly permits a closer association with tertiary structured biomolecules. It is not hygroscopic yet exhibits xe2x80x9cflash solubilityxe2x80x9d on hydration, a property particularly useful for dried vaccines.
The aqueous suspension of the biologically-active material is mixed with a sterile aqueous solution of trehalose and the mixture will be prepared such that it will have a trehalose concentration of from 0.20% to 10% w/v, preferably 2 to 10% and more preferably from 2.5 to 8% w/v. Within the range of trehalose concentrations, the actual concentration used will, in general, depend on the unit size of the biologically-active material. Less trehalose is required for small virus particles than for large cells.
The sterile aqueous trehalose/biologically-active material mixture is subjected to a vacuum drying procedure involving a primary drying step followed by a secondary drying step. Preferably, a conventional freeze drying apparatus (e.g., such as manufactured by EDWARDS, CHRIST, USIFROID or SAVANT) is used for the drying procedure in order to provide a controlled environment for the critical stages in the method. It is emphasised, however, that if such an apparatus is used freeze drying conditions are not employed and the material is not subjected to freeze drying. The drying procedure comprises two drying stages, a primary drying stage in which the residual moisture content of the material is reduced to a value of not greater than 10% and a secondary drying stage in which the residual moisture content of the material is reduced further to a value not exceeding 2%.
In the primary drying stage the initial temperature of the drying apparatus will be such as to ensure that the temperature of the trehalose mixture will not be greater than 37xc2x0 C. and will preferably be 37xc2x0 C. in order to prevent any loss of biological activity at this stage in the method. As water is evaporated off the mixture, the temperature of the mixture falls. The desiccation is, however, carried out to ensure that no freezing or sublimation from ice occurs as is normally experienced in conventional freeze drying procedures, i.e., the temperature of the product during this stage of the drying process is controlled not to fall to 0xc2x0 C. or below and is typically controlled not to fall below 4xc2x0 C. The pressure is reduced below atmospheric pressure, preferably to a value of from 800 to 300 mbar.
The primary drying stage is continued for a period of time, between 30 and 60 minutes, during which time the temperature of the trehalose mixture initially falls as water is evaporated off and then rises. When a temperature (under the reduced pressure employed) of about 25xc2x0 C. is reached during this procedure the trehalose forms a glassy matrix comprising glassy trehalose and containing the biologically-active material which is in a desiccated or partially desiccated state. The primary drying stage is completed when the moisture content of the glassy matrix has reached 10% or below and it is essential, at this critical stage in the procedure, that the temperature is not allowed to exceed 40xc2x0 C. If the temperature at the end of the primary drying stage does exceed 40xc2x0 C. the biologically-active material will sustain damage.
The product obtained from the primary drying stage is then subjected to a secondary drying stage, preferably without removal from the drying apparatus used for the primary drying stage. In the secondary drying stage the glassy matrix obtained from the primary drying stage is further dehydrated under a reduced pressure, not exceeding 0.1 mbar. Currently-available specialised apparatus has the ability to operate at extremely low pressures, for instance below 0.001 mbar. However, very good results have been obtained according to the present invention using reduced pressures for the secondary drying stage in the range of from 0.001 to 0.1 mbar, typically from 0.01 to 0.1 mbar. The secondary drying stage is continued for a total period of time in the range of from 10 to 30 hours, preferably 20 to 30 hours.
As mentioned above, the temperature of the glassy matrix at the end of the primary drying stage does not exceed 40xc2x0 C. During the secondary drying stage the temperature of the matrix may rise slightly to a final temperature, at the end of the drying procedure, of from 40xc2x0 C. to 45xc2x0 C. The trehalose matrix, at the end of the secondary drying stage will have a residual moisture content of 2% or less, preferably 1% or less in order to ensure a very high degree of thermostability in the product. At such residual moisture contents the final temperature in the drying process should not be higher than 45xc2x0 C. since the desiccated biologically-active material in the matrix may undergo some degree of destruction above 45xc2x0 C. Preferably, the temperature of the product at the end of the secondary drying stage will not exceed 44xc2x0 C.
It has been found, through experimentation that there is some benefit in controlling the temperature of the matrix, during the secondary drying stage, such that it is maintained at about 37xc2x0 C. for a period of from 15 to 17 hours and then raising this gradually (for instance by 0.5 to 1xc2x0 C. per hour), over the remaining secondary drying time to a final temperature within the range of 40xc2x0 C. to 45xc2x0 C.
The glassy trehalose matrix produced according to the method can be rehydrated very quickly in an appropriate aqueous medium, typically sterile distilled water, to produce a vaccine for use in a very short period of time.
The preparation of the vaccine and the operating procedure using a freeze dryer for the desiccation of viruses, such as Rinderpest and Peste des Petits Ruminants viruses, and mycoplasma without a lyophilisation step involving sublimination from ice, exploits the unique property of the disaccharide trehalose to protect tertiary macromolecules during desiccation.
Compared to conventional freeze-drying procedures the method of the invention offers the following benefits:
It provides a high level of virus protection, employing a relatively short, simple procedure, e.g., a 25 hour production cycle, thus reducing production cycle time and energy costs.
Basic drying equipment is all that is required, although sophisticated microprocessor controlled freeze dryers can also be used, but are not strictly essential.
The method is tolerant of power interruption, unlike lyophilisation where even a short power failure can cause product meting, leading to unacceptable loss of virus.
Oral vaccination with some attenuated strains of virus has in the past been difficult to achieve because of the loss of epitheliotropism. Both oral and intranasal vaccination would be useful and appropriate for many applications because they mimic the natural route of droplet infection, generating a cascade of protective mucosal immunity, with IgA and humoral IgG2a T helper-cell type 1 response. It would be easy to administer such routes of vaccination and these would be applicable in the event that a suitable vaccine is prepared. A vaccination procedure with live attenuated strains mimicking the natural route of infection induces a more comprehensive sero mucous and cell mediated immunity. According to a further aspect the present invention provides a method of making a vaccine for oral or intranasal use which comprises preparing a glassy matrix of trehalose containing desiccated virus according to the above described method, combined with a suitable positively-charged, biocompatible, water-soluble adjuvant, and rehydrating the glassy matrix with an appropriate aqueous composition. According to a preferred embodiment of this aspect of the invention the vaccine for oral or intranasal vaccination is an MMR vaccine. Since the current paediatric MMR vaccine is prepared by conventional freeze drying technology and is injected into the patient subcutaneously, an oral or intranasal vaccine would give great benefits.